Anaemia is independently associated with mortality in patients with hepatocellular carcinoma

Background Anaemia is frequent in patients with cancer and/or liver cirrhosis and is associated with impaired quality of life. Here, we investigated the impact of anaemia on overall survival (OS) and clinical characteristics in patients with hepatocellular carcinoma (HCC). Materials and methods HCC patients treated between 1992 and 2018 at the Medical University of Vienna were retrospectively analysed. Anaemia was defined as haemoglobin level <13 g/dl in men and <12 g/dl in women. Results Of 1262 assessable patients, 555 (44.0%) had anaemia. The main aetiologies of HCC were alcohol-related liver disease (n = 502; 39.8%) and chronic hepatitis C (n = 375; 29.7%). Anaemia was significantly associated with impaired liver function, portal hypertension, more advanced Barcelona Clinic Liver Cancer stage and elevated C-reactive protein (CRP). In univariable analysis, anaemia was significantly associated with shorter median OS [9.5 months, 95% confidence interval (95% CI) 7.3-11.6 months] versus patients without anaemia (21.5 months, 95% CI 18.3-24.7 months) (P < 0.001). In multivariable analysis adjusted for age, Model for End-stage Liver Disease, number of tumour nodules, size of the largest nodule, macrovascular invasion, extrahepatic spread, first treatment line, alpha-fetoprotein and CRP, anaemia remained an independent predictor of mortality (adjusted hazard ratio 1.23, 95% CI 1.06-1.43, P = 0.006). Conclusions Anaemia was significantly associated with mortality in HCC patients, independent of established liver- and tumour-related prognostic factors. Whether adequate management of anaemia can improve outcome of HCC patients needs further evaluation.


INTRODUCTION
Liver cancer, of which hepatocellular carcinoma (HCC) represents the majority, is the sixth most frequent cancer and the third most common malignant cause of death worldwide. 1Mostly, it develops in a cirrhotic liver due to hepatitis B virus (HBV), hepatitis C virus (HCV), alcohol-related liver disease (ALD) or metabolic dysfunction-associated steatotic liver disease (MASLD). 2 Several prognostic serum biomarkers have been proposed for patients suffering from HCC.While alpha-fetoprotein (AFP) and C-reactive protein (CRP) are amongst the most studied ones, [3][4][5] the prognostic role of anaemia, a condition often found in cancer patients, 6 is less clear.Anaemia is defined by the World Health Organization (WHO) as a haemoglobin level <13 g/dl in men and <12 g/ dl in women. 7It can be stratified by mean corpuscular volume (MCV) into microcytic, normocytic and macrocytic anaemia which is suggestive of specific causes. 8In cancer patients, anaemia is a common comorbidity that is associated with worse outcome and impaired quality of life. 9,10ne important reason for anaemia in malignant diseases is inflammation, leading to anaemia of chronic disease, mainly caused by a dysfunctional iron metabolism. 11urther mechanisms, e.g.chronic bleeding, haemolysis, malnutrition or bone marrow infiltration, may also play a role in certain types of cancer. 9In patients with liver cirrhosis and portal hypertension, anaemia is also frequently present due to various risk factors such as Volume 9 -Issue 6 -2024 https://doi.org/10.1016/j.esmoop.2024.103593][14] Although HCC patients suffer from two conditions commonly accompanied by anaemia (i.e.liver cirrhosis and cancer), direct evidence about the impact of anaemia on their survival is sparse.Two studies found a correlation between low haemoglobin levels and overall survival (OS) in small cohorts of HCC patients. 15,16Low haemoglobin levels were also associated with diminished survival in cohorts treated with programmed cell death protein 1 (PD-1) inhibitors 17 and Yttrium-90 radioembolisation. 18By using computational intelligence methods, a recent study identified haemoglobin levels as one of the most predictive factors for survival of HCC patients although it remains unclear which treatment the patients received. 19ere, we conducted a retrospective analysis of the impact of anaemia on OS and clinical characteristics in a large, well-characterised cohort of HCC patients.

Patients
All adult patients who were diagnosed with HCC at the Medical University of Vienna from 01 August 1992 to 30 April 2018 were included.1][22] Patients with missing haemoglobin level and/or insufficient follow-up were excluded from this analysis.

Data collection and definition of variables
All data were collected retrospectively from the patients' electronic health records.If not stated otherwise, the values at the time of HCC diagnosis were collected.All laboratory parameters recorded for this study were measured in the ISO-certified laboratory of the Medical University of Vienna.ChildePugh score and Model for End-stage Liver Disease (MELD) score were used to describe liver function.Tumour staging was defined according to the Barcelona Clinic Liver Cancer (BCLC) classification. 23The presence of portal hypertension was assumed in all patients with oesophageal/ gastric varices, portal-hypertensive gastropathy, ascites, a hepatic venous pressure gradient of !10 mmHg or a liver stiffness measurement value measured by vibrationcontrolled transient elastography !25 kPa. 24The Milan criteria as a summative parameter for tumour extent were defined according to Mazzaferro et al. 25 (one tumour nodule 5 cm or 3 nodules 3 cm, no macrovascular invasion, no extrahepatic manifestation).
Anaemia was defined according to the WHO definition, 7 i.e. a haemoglobin level below 13 g/dl in men and below 12 g/dl in women.The severity of anaemia was stratified into four grades according to the Common Terminology Criteria of Adverse Events (CTCAE) version 5.0. 26Anaemia type was stratified by MCV into microcytic (<80 fl), normocytic (80-96 fl) and macrocytic anaemia (>96 fl).
The definition of systemic treatment in the context of this manuscript includes sorafenib, lenvatinib, regorafenib, cabozantinib, ramucirumab and the immune checkpoint inhibitors nivolumab, pembrolizumab as well as the combination of atezolizumab and bevacizumab.All other experimental systemic treatments (e.g.thalidomide, octreotide, etc.) were included in the 'other treatment' group.For the purpose of statistical analysis, liver transplantation, surgical resection and local ablation were defined as curative treatments, whereas transarterial chemoembolisation (TACE), systemic therapy, best supportive care (BSC) and all other treatments were defined as palliative treatments.

Definition of endpoints and statistical analysis
Baseline patient characteristics of the overall study population and the subgroups anaemia versus no anaemia are presented using descriptive statistics.The association of categorical variables was tested by chi-square test.Means of continuous variables were compared by t-test.
OS was defined as the time from the initiation of the first treatment line until the date of death.If a patient did not receive any treatment, the date of diagnosis was defined as the starting point of OS.Patients who had still been alive at 31 October 2021 (end of follow-up) were censored at the time of last contact.Median follow-up was calculated by the reverse KaplaneMeier method.
Survival plots were calculated by the KaplaneMeier method and compared by log-rank test (univariable analysis).Multivariable survival analysis was done by Cox regression.Variables were included into the multivariable model if they were statistically significant predictors of OS in univariable analysis.If two or more variables showed a strong correlation (e.g.ChildePugh class and MELD score), only one of these variables was inserted into the multivariable model in order to avoid collinearity.
In general, a P value < 0.05 was considered statistically significant.Wherever appropriate, the level of statistical significance was corrected by using the Bonferroni methoddin this case, the corrected level of statistical significance is given in the footnote of the respective table.Statistical analyses were carried out using SPSS version 29.0 (Chicago, IL) and R 4.3.1 (R Core Team, R Foundation for Statistical Computing, Vienna, Austria).Receiver operating characteristic (ROC) curves for the outcomes of interest were calculated using the 'proc' package of R.

Ethical considerations
This retrospective analysis was approved by the local ethics committee of the Medical University of Vienna (reference number 1759/2015).As this study is a retrospective analysis of anonymised patient data, the obligation to obtain written informed consent was waived by the ethics committee.

Patient characteristics
Of 1415 HCC patients, 152 were a priori excluded from analysis due to missing haemoglobin levels and one because of insufficient follow-up.Thus, 1262 patients were analysed (Supplementary Figure S1, available at https://doi.org/10.1016/j.esmoop.2024.103593).

Association between anaemia and other patient characteristics
At diagnosis, 555 patients (44.0%) had anaemia.Mean haemoglobin level was 12.9 g/dl (SD AE 2.0) in the total cohort, 11.1 g/dl (SD AE 1.3) in patients with anaemia and 14.3 g/dl (SD AE 1.1) in patients without anaemia (Table 2).

DISCUSSION
In this large retrospective study, we identified anaemia as a significant comorbidity in HCC patients which is associated with tumour stage, more advanced liver function impairment and inflammation, and negatively impacts patients' outcome.A key strength of our study is the large number of well-characterised patients with a long follow-up, allowing for meaningful and robust survival analysis.Our results are in line with other, albeit small, studies that have also reported a link between haemoglobin levels and impaired survival in HCC patients treated with PD-1 inhibitors, 17 Yttrium-90 radioembolisation 18 and various first-line treatments. 15,16gure Overall survival (OS) according to the presence or absence of anaemia.
Anaemia is usually stratified by MCV into micro-, normo-and macrocytic anaemia, which is closely linked to possible causes of anaemia. 8,9In our cohort, most anaemic patients suffered from normocytic anaemia, suggesting that inflammation-drivendsocalled 'anaemia of chronic disease' (or more recently termed 'anaemia of inflammation')dwas the most common cause.This is a comorbidity that is frequently found in cancer patients. 11eside tumour stage (i.e.BCLC stage), ChildePugh class, MELD score and portal hypertension were significantly associated with anaemia, underlining that impaired liver function and splenomegaly/hypersplenism due to portal hypertension also contribute to the development of anaemia in HCC patients.Accordingly, in a cohort of patients with advanced chronic liver disease and portal hypertension, most anaemic patients had normocytic anaemia, and the presence of anaemia and lower haemoglobin levels were associated with deteriorating liver function. 12However, given that anaemia and haemoglobin level as a continuous variable were both significantly associated with mortality independent of MELD score in the multivariable model, we conclude that anaemia per se should be considered as an independent risk factor for mortality and not only as a surrogate marker for hepatic dysfunction and more advanced tumour stages.
CRP is a well-known marker for inflammation as well as more aggressive tumour biology and highly predictive for survival and other outcome parameters in HCC patients. 3,4,27,28Its significant association with anaemia in our cohort highlights the well-known and important role of inflammation in the development of anaemia. 11ossible limitations of our study include the retrospective design with all its potential biases and the long period of inclusion, possibly introducing heterogeneity in the diagnostic approach and standard of care.Moreover, some variables required to determine the cause of anaemia (e.g.folic acid, vitamin B12) were not routinely measured.Additionally, we assumed that all patients with ascites had portal hypertension; however, ascites in HCC patients may have other causes (e.g.hypoalbuminaemia, malignant ascites) and thus a small number of patients may have been misclassified as having portal hypertension.
It is noteworthy that cut-offs for haemoglobin levels for clinical decision making (e.g. for clinical trial inclusion, transfusion, erythropoietin administration) [29][30][31] are generally lower than the cut-off defining anaemia according to WHO.In our cohort, we observed no OS difference in patients with mild (CTCAE grade 1) versus moderate/severe (grade 2-4) anaemia, and both groups had a markedly reduced OS compared to non-anaemic HCC patients.Whether targeting anaemia at earlier stages may be beneficial in terms of quality of life and survival of HCC patients requires prospective evaluation.
In conclusion, anaemia was significantly associated with worse OS in HCC patients, independent of various wellknown prognostic factors including severity of liver function impairment.Furthermore, our data suggest a role of more advanced tumour stage, more severe liver function impairment and systemic inflammation in the multifactorial genesis of anaemia in patients with HCC.Prospective studies are needed to investigate potential benefits of measures to ameliorate anaemia in patients with HCC.

FUNDING
None declared.

DISCLOSURE
TM received speaker honoraria from AstraZeneca, Chiesi and Janssen-Cilag; consulting fees from CSL Behring; and travel support from AstraZeneca, BeiGene, CSL Behring, Chiesi, Jazz Pharmaceuticals, Janssen-Cilag, Novartis and tevaratiopharm.LBa received speaker honoraria from Chiesi.MM served as a speaker and/or consultant and/or advisory board member for AbbVie, Collective Acumen, Gilead and W.

Table 1 .
Patient characteristics